Real Time Cleavage Assay

ABSTRACT

A cleavage-based real-time PCR assay method is provided. In general terms, the assay method includes subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target, and b) flap cleavage reagents for performing a flap cleavage assay on the amplified nucleic acid target to two sets of thermocycling conditions. No additional reagents are added to the reaction between said first and second sets of cycles and, in each cycle of the second set of cycles, cleavage of a flap probe is measured.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/946,737, filed on Nov. 10, 2010, all of which isincorporated by reference in its entirety.

BACKGROUND

Several point mutations in the human genome have a direct associationwith a disease. For example, several germline KRAS mutations have beenfound to be associated with Noonan syndrome (Schubbert et al. Nat.Genet. 2006 38: 331-6) and cardio-facio-cutaneous syndrome (Niihori etal. Nat. Genet. 2006 38: 294-6). Likewise, somatic KRAS mutations arefound at high rates in leukemias, colorectal cancer (Burmer et al. Proc.Natl. Acad. Sci. 1989 86: 2403-7), pancreatic cancer (Almoguera et al.Cell 1988 53: 549-54) and lung cancer (Tam et al. Clin. Cancer Res. 200612: 1647-53). Many point mutations in the human genome have no apparentcausative association with a disease.

Methods for the detection of point mutations may be used, for example,to provide a diagnostic for diseases that are associated with the pointmutations.

SUMMARY

A cleavage-based real-time PCR assay method is provided. In certainembodiments, the assay method includes subjecting a reaction mixturecomprising a) PCR reagents for amplifying a nucleic acid target, and b)flap cleavage reagents for performing a flap cleavage assay on theamplified nucleic acid target to two sets of thermocycling conditions.The first set of thermocycling conditions includes a set of 5-15 cyclesof: i. a first temperature of at least 90° C.; ii. a second temperaturein the range of 60° C. to 75° C.; iii. a third temperature in the rangeof 65° C. to 75° C. The second and third temperatures may be the same.The second set of thermocycling conditions includes a set of 20-50cycles of: i. a fourth temperature of at least 90° C.; ii. a fifthtemperature that is at least 10° C. lower than the second temperature;iii. a sixth temperature in the range of 65° C. to 75° C. In certaincases, no additional reagents are added to the reaction between thefirst and second sets of cycles and, in each cycle of the second set ofcycles, cleavage of a flap probe is measured.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates some of the general principles of aflap assay.

FIG. 2 shows results of an assay done using single stage thermocycling.Detection and quantitation of the KRAS G35T mutation in the presence ofthe wild type sequence at levels, as indicated. Kinetic curves show allratios of mutant to wild type other than 1:10 and 1:100 areindistinguishable.

FIG. 3 shows results of an assay done using two stage thermocycling.Detection and quantitation of the KRAS G35T mutation in the presence ofthe wild type sequence at levels, as indicated. Kinetic curves showresolution of ratios from 1:10 to 1:10,000.

DEFINITIONS

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in liquid form,containing one or more analytes of interest.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the likes.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotidesor ribonucleotides, and may be produced enzymatically or synthetically(e.g., PNA as described in U.S. Pat. No. 5,948,902 and the referencescited therein) which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. Naturally-occurring nucleotides include guanine,cytosine, adenine and thymine (G, C, A and T, respectively).

The term “nucleic acid sample,” as used herein denotes a samplecontaining nucleic acid.

The term “target polynucleotide,” as used herein, refers to apolynucleotide of interest under study. In certain embodiments, a targetpolynucleotide contains one or more target sites that are of interestunder study.

The term “oligonucleotide” as used herein denotes a single strandedmultimer of nucleotides of about 2 to 200 nucleotides. Oligonucleotidesmay be synthetic or may be made enzymatically, and, in some embodiments,are 10 to 50 nucleotides in length. Oligonucleotides may containribonucleotide monomers (i.e., may be oligoribonucleotides) ordeoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 11 to30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to150 or 150 to 200 nucleotides in length, for example.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotides that are base-paired, i.e., hybridizedtogether.

The term “primer” as used herein refers to an oligonucleotide that has anucleotide sequence that is complementary to a region of a targetpolynucleotide. A primer binds to the complementary region and isextended, using the target nucleic acid as the template, under primerextension conditions. A primer may be in the range of about 15 to about50 nucleotides although primers outside of this length may be used. Aprimer can be extended from its 3′ end by the action of a polymerase. Anoligonucleotide that cannot be extended from it 3′ end by the action ofa polymerase is not a primer.

The term “extending” as used herein refers to any addition of one ormore nucleotides to the end of a nucleic acid, e.g. by ligation of anoligonucleotide or by using a polymerase.

The term “amplifying” as used herein refers to generating one or morecopies of a target nucleic acid, using the target nucleic acid as atemplate.

The term “denaturing,” as used herein, refers to the separation of anucleic acid duplex into two single strands.

The terms “determining”, “measuring”, “evaluating”, “assessing,”“assaying,” “detecting,” and “analyzing” are used interchangeably hereinto refer to any form of measurement, and include determining if anelement is present or not. These terms include both quantitative and/orqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

The term “using” has its conventional meaning, and, as such, meansemploying, e.g., putting into service, a method or composition to attainan end.

As used herein, the term “T_(m)” refers to the melting temperature of anoligonucleotide duplex at which half of the duplexes remain hybridizedand half of the duplexes dissociate into single strands. The T_(m) of anoligonucleotide duplex may be experimentally determined or predictedusing the following formula T_(m)=81.5+16.6(log₁₀[Na⁺])+0.41 (fractionG+C)−(60/N), where N is the chain length and [Na⁺] is less than 1 M. SeeSambrook and Russell (2001; Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., ch. 10).Other formulas for predicting T_(m) of oligonucleotide duplexes existand one formula may be more or less appropriate for a given condition orset of conditions.

As used herein, the term “T_(m)-matched” refers to a plurality ofnucleic acid duplexes having T_(m)s that are within a defined range,e.g., within 5° C. or 10° C. of each other.

As used herein, the term “reaction mixture” refers to a mixture ofreagents that are capable of reacting together to produce a product inappropriate external conditions over a period of time. A reactionmixture may contain PCR reagents and flap cleavage reagents, forexample, the recipes for which are independently known in the art.

The term “mixture”, as used herein, refers to a combination of elements,that are interspersed and not in any particular order. A mixture isheterogeneous and not spatially separable into its differentconstituents. Examples of mixtures of elements include a number ofdifferent elements that are dissolved in the same aqueous solution, or anumber of different elements attached to a solid support at random or inno particular order in which the different elements are not speciallydistinct. A mixture is not addressable. To illustrate by example, anarray of spatially separated surface-bound polynucleotides, as iscommonly known in the art, is not a mixture of surface-boundpolynucleotides because the species of surface-bound polynucleotides arespatially distinct and the array is addressable.

As used herein, the term “PCR reagents” refers to all reagents that arerequired for performing a polymerase chain reaction (PCR) on a template.As is known in the art, PCR reagents essentially include a first primer,a second primer, a thermostable polymerase, and nucleotides. Dependingon the polymerase used, ions (e.g., Mg²⁺) may also be present. PCRreagents may optionally contain a template from which a target sequencecan be amplified.

As used herein, the term “flap assay” refers to an assay in which a flapoligonucleotide is cleaved in an overlap-dependent manner by a flapendonuclease to release a flap that is then detected. The principles offlap assays are well known and described in, e.g., Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447). Forthe sake of clarity, certain reagents that are employed in a flap assayare described below. The principles of a flap assay are illustrated inFIG. 1. In the flap assay shown in FIG. 1, an invasive oligonucleotide 2and flap oligonucleotide 4 are hybridized to target 6 to produce a firstcomplex 8 that contains a nucleotide overlap at position 10. Firstcomplex 8 is a substrate for flap endonuclease. Flap endonuclease 12cleaves flap oligonucleotide 4 to release a flap 14 that hybridizes withFRET cassette 16 that contains a quencher “Q” and a nearby quenchedflourophore “R” that is quenched by the quencher Q. Hybridization offlap 14 to FRET cassette 16 results in a second complex 18 that containsa nucleotide overlap at position 20. The second complex is also asubstrate for flap endonuclease. Cleavage of FRET cassette 16 by flapendonuclease 12 results in release of the fluorophore 22, which producesa fluorescent signal. These components are described in greater detailbelow.

As used herein, the term “invasive oligonucleotide” refers to anoligonucleotide that is complementary to a region in a target nucleicacid. The 3′ terminal nucleotide of the invasive oligonucleotide may ormay not base pair a nucleotide in the target (e.g., which may be thesite of a SNP or a mutation, for example).

As used herein, the term “flap oligonucleotide” refers to anoligonucleotide that contains a flap region and a region that iscomplementary to a region in the target nucleic acid. The targetcomplementary regions on the invasive oligonucleotide and the flapoligonucleotide overlap by a single nucleotide. As is known, if the 3′terminal nucleotide of the invasive nucleotide and the nucleotide thatoverlaps that nucleotide in the flap oligonucleotide both base pair witha nucleotide in the target nucleic acid, then a particular structure isformed. This structure is a substrate for an enzyme, defined below as aflap endonuclease, that cleaves the flap from the target complementaryregion of the flap oligonucleotide. If the 3′ terminal nucleotide of theinvasive oligonucleotide does not base pair with a nucleotide in thetarget nucleic acid, or if the overlap nucleotide in the flapoligononucleotide does not base pair with a nucleotide in the targetnucleic acid, the complex is not a substrate for the enzyme and there islittle or no cleavage.

The term “flap endonuclease” or “FEN” for short, as used herein, refersto a class of nucleolytic enzymes that act as structure specificendonucleases on DNA structures with a duplex containing a singlestranded 5′ overhang, or flap, on one of the strands that is displacedby another strand of nucleic acid, i.e., such that there are overlappingnucleotides at the junction between the single and double-stranded DNA.FENs catalyze hydrolytic cleavage of the phosphodiester bond at thejunction of single and double stranded DNA, releasing the overhang, orthe flap. Flap endonucleases are reviewed by Ceska and Savers (TrendsBiochem. Sci. 1998 23:331-336) and Liu et al (Annu. Rev. Biochem. 200473: 589-615). FENs may be individual enzymes, multi-subunit enzymes, ormay exist as an activity of another enzyme or protein complex, e.g., aDNA polymerase. A flap endonuclease may be thermostable.

As used herein, the term “cleaved flap” refers to a single-strandedoligonucleotide that is a cleavage product of a flap assay.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleaved flapwith a FRET cassette produces a secondary substrate for the flapendonuclease. Once this substrate is formed, the 5′fluorophore-containing base is cleaved from the cassette, therebygenerating a fluorescence signal.

As used herein, the term “flap assay reagents” refers to all reagentsthat are required for performing a flap assay on a substrate. As isknown in the art, flap assays include an invasive oligonucleotide, aflap oligonucleotide, a flap endonuclease and a FRET cassette, asdescribed above. Flap assay reagents may optionally contain a target towhich the invasive oligonucleotide and flap oligonucleotide bind.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Described herein is a cleavage-based real-time PCR assay method. Ingeneral terms, the assay method includes subjecting a reaction mixturecomprising a) PCR reagents for amplifying a nucleic acid target, and b)flap cleavage reagents for performing a flap cleavage assay on theamplified nucleic acid target to two sets of thermocycling conditions.In certain cases, no additional reagents are added to the reactionbetween the first and second sets of cycles and, in each cycle of thesecond set of cycles, cleavage of a flap probe is measured. In furtherdescribing the method, the reagent mixture used in the method will bedescribed first, followed by a description of the thermocyclingconditions used in the method.

In the following description, the skilled artisan will understand thatany of a number of polymerases and flap endonucleases could be used inthe methods, including without limitation, those isolated fromthermostable or hyperthermostable prokaryotic, eukaryotic, or archaealorganisms. The skilled artisan will also understand that the enzymesthat are used in the method, e.g., polymerase and flap endonuclease,include not only naturally occurring enzymes, but also recombinantenzymes that include enzymatically active fragments, cleavage products,mutants, and variants of wild type enzymes.

Reaction Mixture

As noted above, the reaction mixture used in the method contains atleast PCR reagents for amplifying a nucleic acid target and flapcleavage reagents for performing a flap cleavage assay on the amplifiednucleic acid. The reaction mixture employed in the method may thereforecontain a pair of primers as well a reaction buffer (which can be pHbuffered and may include salt, e.g., MgCl₂ and other componentsnecessary for PCR), nucleotides, e.g., dGTP, dATP, dTTP and dCTP and athermostable DNA polymerase, as well as a flap oligonucleotide, a flapendonuclease and a FRET cassette, as defined above. Depending on how theassay is performed (i.e., depending on whether one of the PCR primers isused as an invasive oligonucleotide in the flap assay) the reaction mixmay additionally contain an invasive oligonucleotide that is distinctfrom the PCR primers. The reaction mixture may further contain a nucleicacid target.

The exact identities and concentrations of the reagents present in thereaction mixture may be similar to or the same as those independentlyemployed in PCR and flap cleavage assays, with the exception that thereaction mixture contains Mg²⁺ at a concentration that is higher thenemployed in conventional PCR reaction mixtures (which contain Mg²⁺ at aconcentration of between about 1.8 mM and 3 mM). In certain embodiments,the reaction mixture described herein contains Mg²⁺ at a concentrationin the range of 4 mM to 10 mM, e.g., 6 mM to 9 mM. Exemplary reactionbuffers and DNA polymerases that may be employed in the subject reactionmixture include those described in various publications (e.g., Ausubel,et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995and Sambrook et al., Molecular Cloning: A Laboratory Manual, ThirdEdition, 2001 Cold Spring Harbor, N.Y.). Reaction buffers and DNApolymerases suitable for PCR may be purchased from a variety ofsuppliers, e.g., Invitrogen (Carlsbad, Calif.), Qiagen (Valencia,Calif.) and Stratagene (La Jolla, Calif.). Exemplary polymerases includeTaq, Pfu, Pwo, UlTma and Vent, although many other polymerases may beemployed in certain embodiments. Guidance for the reaction componentssuitable for use with a polymerase as well as suitable conditions fortheir use, is found in the literature supplied with the polymerase.Primer design is described in a variety of publications, e.g.,Diffenbach and Dveksler (PCR Primer, A Laboratory Manual, Cold SpringHarbor Press 1995); R. Rapley, (The Nucleic Acid Protocols Handbook(2000), Humana Press, Totowa, N.J.); Schena and Kwok et al., Nucl. AcidRes. 1990 18:999-1005). Primer and probe design software programs arealso commercially available, including without limitation, PrimerDetective (ClonTech, Palo Alto, Calif.), Lasergene, (DNASTAR, Inc.,Madison, Wis.); and Oligo software (National Biosciences, Inc.,Plymouth, Minn.) and iOligo (Caesar Software, Portsmouth, N.H).

Exemplary flap cleavage assay reagents are found in Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447).Appropriate conditions for flap endonuclease reactions are either knownor can be readily determined using methods known in the art (see, e.g.,Kaiser et al., J. Biol. Chem. 274:21387-94, 1999). Exemplary flapendonucleases that may be used the method include, without limitation,Thermus aquaticus DNA polymerase I, Thermus thermophilus DNA polymeraseI, mammalian FEN-1, Archaeoglobus fulgidus FEN-1, Methanococcusjannaschii FEN-1, Pyrococcus furiosus FEN-1, Methanobacteriumthermoautotrophicum FEN-1, Thermus thermophilus FEN-1, CLEAVASE™ (ThirdWave, Inc., Madison, Wis.), S. cerevisiae RTH1, S. cerevisiae RAD27,Schizosaccharomyces pombe rad2, bacteriophage T5 5′-3′ exonuclease,Pyroccus horikoshii FEN-1, human exonuclease 1, calf thymus 5′-3′exonuclease, including homologs thereof in eubacteria, eukaryotes, andarchaea, such as members of the class II family of structure-specificenzymes, as well as enzymatically active mutants or variants thereof.Descriptions of cleaving enzymes can be found in, among other places,Lyamichev et al., Science 260:778-83, 1993; Eis et al., Nat. Biotechnol.19:673-76, 2001; Shen et al., Trends in Bio. Sci. 23:171-73, 1998;Kaiser et al. J. Biol. Chem. 274:21387-94, 1999; Ma et al., J. Biol.Chem. 275:24693-700, 2000; Allawi et al., J. Mol. Biol. 328:537-54,2003; Sharma et al., J. Biol. Chem. 278:23487-96, 2003; and Feng et al.,Nat. Struct. Mol. Biol. 11:450-56, 2004.

In particular embodiments, the reaction mix may contain reagents forassaying multiple (e.g., at least 2, 3, 4 or more) different targetssequences in parallel. In these cases, the reaction mix may containmultiple pairs of PCR primers, multiple different flap oligonucleotideshaving different flaps, and multiple different FRET cassettes fordetecting the different flaps, once they are cleaved. In one embodiment,oligonucleotides in a mixture may have common flaps but differentbinding sequences to allow for, for example, a set of mutations tocleave a common FRET cassette and report a signal where a singlefluorophore is indicative of the presence of a mutation. In thisembodiment, which mutation is present in the sample may be determinedafter the presence of a mutation has identified. Optionally, thereaction may contain multiple invasive oligonucleotides if one of thePCR primers is not used as an invasive oligonucleotide. Upon cleavage ofthe FRET cassettes, multiple distinguishable fluorescent signals may beobserved. The fluorophore may be selected from, e.g.,6-carboxyfluorescein (FAM), which has excitation and emissionwavelengths of 485 nm and 520 nm respectively, Redmond Red, which hasexcitation and emission wavelengths of 578 nm and 650 nm respectivelyand Yakima Yellow, which has excitation and emission wavelengths of 532nm and 569 nm respectively, and Quasor670 which has excitation andemission wavelengths of 644 nm and 670 nm respectively, although manyothers could be employed. In certain cases, at least one of the PCRprimer pairs, flap oligonucleotides and FRET cassettes may be for thedetection of an internal control.

As would be apparent, the various oligonucleotides used in the methodare designed so as to not interfere with each other. For example, inparticular embodiments, the flap oligonucleotide may be capped at its 3′end, thereby preventing its extension. Likewise, in certain embodiments,the invasive oligonucleotide may also be capped at its 3′ end if it isnot used as one of the PCR primers. In particular embodiment, if theinvasive oligonucleotide is not used as one of the PCR primers, then theinvasive oligonucleotide may be present at a concentration that is inthe range of 5% to 50%, e.g., 10% to 40% of the concentration of the PCRprimers. Further, in certain cases, the T_(m)s of the flap portion andthe target complementary regions of the flap oligonucleotide mayindependently be at least 10° C. lower (e.g., 10-20° C. lower) than theT_(m)s of the PCR primers, which results in a) less hybridization of theflap oligonucleotide to the target nucleic acid at higher temperatures(60° C. to 75° C.) and b) less hybridization of any cleaved clap to theFRET cassette at higher temperatures (60° C. to 75° C.). The lower fifthtemperature is favorable for hybridization of the oligonucleotides usedin the flap assay, and for the activity of the flap endonuclease.

In a multiplex reaction, the primers may be designed to have similarthermodynamic properties, e.g., similar T_(m)s, G/C content, hairpinstability, and in certain embodiments may all be of a similar length,e.g., from 18 to 30 nt, e.g., 20 to 25 nt in length. The other reagentsused in the reaction mixture may also be T_(m) matched.

The assay mixture may be present in a vessel, including withoutlimitation, a tube; a multi-well plate, such as a 96-well, a 384-well, a1536-well plate; and a microfluidic device. In certain embodiments,multiple multiplex reactions are performed in the same reaction vessel.Depending on how the reaction is performed, the reaction mixture may beof a volume of 5 μl to 200 μl, e.g., 10 μl to 100 μl, although volumesoutside of this range are envisioned.

In certain embodiments, a subject reaction mix may further contain anucleic acid sample. In particular embodiments, the sample may containgenomic DNA or an amplified version thereof (e.g., genomic DNA amplifiedusing the methods of Lage et al, Genome Res. 2003 13: 294-307 orpublished patent application US20040241658, for example). In exemplaryembodiments, the genomic sample may contain genomic DNA from a mammaliancell such a human, mouse, rat or monkey cell. The sample may be madefrom cultured cells or cells of a clinical sample, e.g., a tissuebiopsy, scrape or lavage or cells of a forensic sample (i.e., cells of asample collected at a crime scene). In particular embodiments, thegenomic sample may be from a formalin fixed paraffin embedded (FFPE)sample.

In particular embodiments, the nucleic acid sample may be obtained froma biological sample such as cells, tissues, bodily fluids, and stool.Bodily fluids of interest include but are not limited to, blood, serum,plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid,tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovialfluid, urine, amniotic fluid, and semen. In particular embodiments, asample may be obtained from a subject, e.g., a human, and it may beprocessed prior to use in the subject assay. For example, the nucleicacid may be extracted from the sample prior to use, methods for whichare known.

For example, DNA can be extracted from stool from any number ofdifferent methods, including those described in, e.g, Coll et al (J. ofClinical Microbiology 1989 27: 2245-2248), Sidransky et al (Science 1992256: 102-105), Villa (Gastroenterology 1996 110: 1346-1353) and Nollau(BioTechniques 1996 20: 784-788), and U.S. Pat. Nos. 5,463,782,7,005,266, 6,303,304 and 5,741,650. Commercial DNA extraction kits forthe extraction of DNA from stool include the QIAamp stool mini kit(QIAGEN, Hilden, Germany), Instagene Matrix (Bio-Rad, Hercules, Calif.),and RapidPrep Micro Genomic DNA isolation kit (Pharmacia Biotech Inc.Piscataway, N.J.), among others.

Method for Sample Analysis

In performing the subject method, the reaction mixture is generallysubjected to the following thermocycling conditions: a first set of 5 to15 (e.g., 8 to 12) cycles of: i. a first temperature of at least 90° C.;ii. a second temperature in the range of 60° C. to 75° C. (e.g., 65° C.to 75° C.); iii. a third temperature in the range of 65° C. to 75° C.;followed by: a second set of 20-50 cycles of: i. a fourth temperature ofat least 90° C.; ii. a fifth temperature that is at least 10° C. lowerthan the second temperature (e.g., in the range of 50° C. to 55° C.; andiii. a sixth temperature in the range of 65° C. to 75° C. No additionalreagents need to be added to the reaction mixture during thethermocycling, e.g., between the first and second sets of cycles. Inparticular embodiments, the thermostable polymerase is not inactivatedbetween the first and second sets of conditions, thereby allowing thetarget to be amplified during each cycle of the second set of cycles. Inparticular embodiments, the second and third temperatures are the sametemperature such that “two step” theremocycling conditions areperformed. Each of the cycles may be independently of a duration in therange of 10 seconds to 3 minutes, although durations outside of thisrange are readily employed.

In each cycle of the second set of cycles (e.g., while the reaction isin the fifth temperature), a signal generated by cleavage of the flapprobe may be measured to provide a real-time measurement of the amountof target nucleic acid in the sample (where the term “real-time” isintended to refer to a measurement that is taken as the reactionprogresses and products accumulate). The measurement may be expressed asan absolute number of copies or a relative amount when normalized to acontrol nucleic acid in the sample.

Without being bound to any specific theory, it is believed that thehigher reaction temperatures in the first set of cycles may allow thetarget nucleic acid to be efficiently amplified by the pair of PCRprimers without significant interference by any of the flap assayreagents or their reaction products. The lower reaction temperature usedin the second set of cycles (i.e., the fifth temperature) is not optimumfor the polymerase used for PCR, but allows the flap oligonucleotide toefficiently hybridize to the target nucleic acid and is closer to theoptimum temperature of the flap endonuclease. The lower reactiontemperature used in the second set of cycles also facilitates subsequenthybridization of the cleaved flap to the FRET cassette. Thus, at a lowertemperature, the target nucleic acid may be detected without significantinterference from the PCR reagents.

In certain cases, fluorescence indicating the amount of cleaved flap canbe detected by an automated fluorometer designed to perform real-timePCR having the following features: a light source for exciting thefluorophore of the FRET cassette, a system for heating and coolingreaction mixtures and a fluorometer for measuring fluorescence by theFRET cassette. This combination of features, allows real-timemeasurement of the cleaved flap, thereby allowing the amount of targetnucleic acid in the sample to be quantified. Automated fluorometers forperforming real-time PCR reactions are known in the art and can beadapted for use in this specific assay, for example, the ICYCLER™ fromBio-Rad Laboratories (Hercules, Calif.), the Mx3000P™, the MX3005P™ andthe MX4000™ from Stratagene (La Jolla, Calif.), the ABI PRISM™ 7300,7500, 7700, and 7900 Taq Man (Applied Biosystems, Foster City, Calif.),the SMARTCYCLER™, ROTORGENE 2000™ (Corbett Research, Sydney, Australia)and the GENE XPERT™ System (Cepheid, Sunnyvale, Calif.) and theLIGHTCYCLER™ (Roche Diagnostics Corp., Indianapolis, Ind.). The speed oframping between the different reaction temperatures is not critical and,in certain embodiments, the default ramping speeds that are preset onthermocyclers may be employed.

In certain cases, the method may further involve graphing the amount ofcleavage that occurs at each of the second set of cycles, therebyproviding an estimate of the abundance of the nucleic acid target. Theestimate may be calculated by determining the threshold cycle (i.e., thecycle at which this fluorescence increases above a predeterminedthreshold; the “Ct” value or “Cp” value). This estimate can be comparedto a control (which control may be assayed in the same reaction mix asthe genomic locus of interest) to provide a normalized estimate. Thethermocycler may also contain a software application for determining thethreshold cycle for each of the samples. An exemplary method fordetermining the threshold cycle is set forth in, e.g., Luu-The et al(Biotechniques 2005 38: 287-293).

A device for performing sample analysis is also provided. In certainembodiments, the device comprises: a) a thermocycler programmed toperform the above-described and b) a vessel comprising: PCR reagents foramplifying a nucleic acid target, and flap cleavage reagents forperforming a flap cleavage assay on the nucleic acid target.

Utility

The method described finds use in a variety of applications, where suchapplications generally include sample analysis applications in which thepresence of a target nucleic acid sequence in a given sample isdetected.

In particular, the above-described methods may be employed to diagnose,to predict a response to treatment, or to investigate a cancerouscondition or another mammalian disease, including but not limited to,leukemia, breast carcinoma, prostate cancer, Alzheimer's disease,Parkinsons's disease, epilepsy, amylotrophic lateral schlerosis,multiple sclerosis, stroke, autism, mental retardation, anddevelopmental disorders. Many nucleotide polymorphisms are associatedwith and are thought to be a factor in producing these disorders.Knowing the type and the location of the nucleotide polymorphism maygreatly aid the diagnosis, prognosis, and understanding of variousmammalian diseases. In addition, the assay conditions described hereincan be employed in other nucleic acid detection applications including,for example, for the detection of infectious diseases, viral loadmonitoring, viral genotyping, environmental testing, food testing,forensics, epidemiology, and other areas where specific nucleic acidsequence detection is of use.

In some embodiments, a biological sample may be obtained from a patient,and the sample may be analyzed using the method. In particularembodiments, the method may be employed to identify and/or estimate theamount of mutant copies of a genomic locus that are in a biologicalsample that contains both wild type copies of a genomic locus and mutantcopies of the genomic locus that have a point mutation relative to thewild type copies of the genomic locus. In this example, the sample maycontain at least 100 times (e.g., at least 1,000 times, at least 5,000times, at least 10,000 times, at least 50,000 times or at least 100,000times) more wild type copies of the genomic locus than mutant copiessaid genomic locus.

In these embodiments, the method may be employed to detect an oncogenicmutation (which may be a somatic mutation) in, e.g., PIK3CA, NRAS, KRAS,JAK2, HRAS, FGFR3, FGFR1, EGFR, CDK4, BRAF, RET, PGDFRA, KIT or ERBB2,which mutation may be associated with breast cancer, melanoma, renalcancer, endometrial cancer, ovarian cancer, pancreatic cancer, leukemia,colorectal cancer, prostate cancer, mesothelioma, glioma, meullobastoma,polythemia, lymphoma, sarcoma or multiple myeloma (see, e.g., Chial 2008Proto-oncogenes to oncogenes to cancer. Nature Education 1:1).

In these embodiments, the reaction mixture may contain a first primerand a second primer wherein the first primer comprises a 3′ terminalnucleotide that base pairs with the point mutation. The first primer maybe employed as the invasive oligonucleotide in the second set of cyclesor, in certain cases, there may be a separate invasive oligonucleotidepresent in the reaction mixture that also has a 3′ terminal nucleotidethat base pairs with the point mutation. Since the point mutation in thegenomic locus may have a direct association with cancer, e.g.,colorectal cancer, the subject method may be employed to diagnosepatients with cancer, alone, or in combination with other clinicaltechniques (e.g., a physical examination such as a colonoscopy orimmunohistochemical analysis) or molecular techniques. For example,results obtained from the subject assay may be combined with otherinformation, e.g., information regarding the methylation status of otherloci, information regarding in the same locus or at a different locus,cytogenetic information, information regarding rearrangements, geneexpression information or information about the length of telemerers, toprovide an overall diagnosis of cancer or other diseases.

In one embodiment, a sample may be collected from a patient at a firstlocation, e.g., in a clinical setting such as in a hospital or at adoctor's office, and the sample may forwarded to a second location,e.g., a laboratory where it is processed and the above-described methodis performed to generate a report. A “report” as described herein, is anelectronic or tangible document which includes report elements thatprovide test results that may include a Ct or Cp value or the like thatindicates the presence of mutant copies of the genomic locus in thesample. Once generated, the report may be forwarded to another location(which may the same location as the first location), where it may beinterpreted by a health professional (e.g., a clinician, a laboratorytechnician, or a physician such as an oncologist, surgeon, pathologist),as part of a clinical diagnosis.

Kits

Also provided are kits for practicing the subject method, as describedabove. The components of the kit may be present in separate containers,or multiple components may be present in a single container.

In addition to above-mentioned components, the kit may further includeinstructions for using the components of the kit to practice the subjectmethods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

In addition to the instructions, the kits may also include one or morecontrol samples, e.g., positive or negative controls analytes for use intesting the kit.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Example 1 KRAS G35T Assay

The assay described below is designed to detect nucleic acid sequencescontaining the KRAS G35T mutation in a background of wild typesequences. For reference, partial nucleotide sequences for the wild typeand G35T mutant alleles of KRAS are shown below.

Partial sequence of amplification region for KRAS, wild type (position35 underlined):

(SEQ ID NO: 1) ATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGG 

Partial sequence of amplification region for KRAS, mutant G35T (position35 underlined):

(SEQ ID NO: 2) ATGACTGAATATAAACTTGTGGTAGTTGGAGCTGTTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGG 

The ability to detect the KRAS mutation T at position 35 in a backgroundof wild type G at position 35 was tested using two differentthermocycling protocols, one of which uses single stage cycling and theother uses two stage cycling (see Table 1). In both protocols, at alldilutions, approximately 100,000 copies (i.e., 100,000 double strandedplasmids) of the wild type sequence were present. To the 100,000 copiesof wild type, approximately 10,000, 1,000, 100, and 10 copies of themutant target gene were added. A sample containing 100,000 copies of themutant sequence was used as a control.

Table 1 summarizes the cycling conditions for the cleavage-based assayfor single stage thermocycling and two stage thermocycling. Fluorescentsignal acquisition occurs at the 53° C. temperature, conducive to thecleavage reaction of the flap probe from the target and the cleavage ofthe fluorophore from the FRET cassette as mediated by the released flap.

TABLE 1 Single Stage Cycling Compared to 2-Stage (Headstart) ProtocolFluo- Single 2-Stage rescent Stage: (Headstart): Signal Temper- Numberof Number of Acqui- Stage ature Time Cycles Cycles sition Pre-incubation95° C.   2 min. 1 1 None (enzyme activation) Amplification 95° C. 20sec. NONE 10 None (Pre-Amp, 67° C. 30 sec. None Headstart) 70° C. 30sec. None Amplification 95° C. 20 sec  50 45 None 53° C.   1 min. Single70° C. 30 sec. None Cooling (Hold) 40° C. 30 sec. 1 1 None

Primers for the PCR amplification of the KRAS G35T mutation were5′-CTATTGT TGGATCATATTCGTC-3′ (SEQ ID NO:3) as the reverse primer and5′-ACTTGTGGTAGT TGGAGCTCT-3′ (SEQ ID NO:4) as the forward primer. Notethat in the forward primer, the 3′T base (underlined) corresponds to themutant position 35. The penultimate C at position 34 is also a mismatchto both the mutant and wild type sequence, and is designed to increasethe discrimination of the 3′ base against the wild type target.

The homogeneous detection of the KRAS G35T mutation was accomplished bythe use of an endonuclease cleavable flap probe, a cleavable FRETcassette, and a heat stable flap endonuclease. For the detection of theG35T mutation, the flap probe sequence was5′-GACGCGGAGTTGGCGTAGGCA-3′/3C6 (SEQ ID NO:5), where the mutant base isshown underlined and the 3′-end is blocked with a hexanediol group inorder to inhibit extension. The cleaved flap portion, which subsequentlybinds the FRET cassette, and in turn releases the fluorophore away fromits quencher, includes all of the bases from the 5′-end to themutation-specific T. Primers and flap probes were supplied asnon-catalog items by Integrated DNA Technologies (IDT, Coralville,Iowa).

The FRET cassette used was 5′-FAM/TCT/Quencher/AGCCGGTTTTCCGGCTGAGACTCCGCGTCCGT-3′/3C6 (SEQ ID NO:6), where FAM is fluorescein, thequencher is the Eclipse® Dark Quencher, and the 3′-end is blocked with ahexanediol group in order to inhibit primer extension. The FRET cassettewas supplied by Hologic (Madison, Wis.).

The PCR reactions were done in LightCycler® 480 Multiwell 96 Plates(Roche, Indianapolis) in 10 mM MOPS pH 7.5, with 7.5 mM MgCl₂, and 250μM dNTPs (Promega, Madison, Wis.). Taq polymerase was the iTaq enzyme(BioRad, Hercules, Calif.) and the cleavage enzyme was Cleavase 2.0(Hologic, Madison, Wis.). Forward primer concentration was 50 nM,reverse primer concentration was 500 nM, flap probe was at 500 nM, andthe FRET cassette was used at a final concentration of 200 nM. Allamplification and detection was performed in the LightCycler 480 opticalthermocycler (Roche, Indianapolis, Ind.).

Raw data and kinetic curves, as generated by the LightCycler, for thetwo different cycling conditions, as summarized in Table 1, are shown inFIGS. 2 and 3. The results, showing the improved linear quantitativeresponse of the 2-stage cycling protocol are delineated in Table 2.

Table 2 shows the detection and quantitation of the KRAS G35T mutationin the presence of the wild type sequence at levels, as indicated,comparing two different cycling protocols. The point at which thefluorescence of a sample rises above the background fluorescence iscalled the “crossing point (Cp)” of the sample (Roche LightCycler 480Manual, Indianapolis, Ind.), and in these assays is calculated as beingthe point at which fluorescence rose to 18% of the maximum fluorescence.Cp levels above 40 cycles show no detectable dose response.

TABLE 2 detection and quantitation of the KRAS G35T mutation 2-StageWild Ratio of 1-Stage (Headstart) Mutant 35T Type 35G Mutant:WildCrossing Point Crossing Point copies copies Type (Cp) (Cp) 0 100000 N/A44.56 40.33 0 100000 N/A 43.87 40.27 10 99990  1:10000 43.99 38.89 1099990  1:10000 43.04 38.09 100 99900 1:1000 43.39 36.59 100 99900 1:100043.66 36.31 1000 99000 1:100  43.66 31.41 1000 99000 1:100  39.70 31.8210000 90000 1:10  39.62 26.71 10000 90000 1:10  33.68 26.73 100000 0 N/A27.72 20.62 100000 0 N/A 28.04 20.45

1-13. (canceled)
 14. A method of sample analysis comprising: (a)subjecting a reaction mixture comprising: a target nucleic acid; PCRreagents for amplifying the target nucleic acid, and flap cleavagereagents for performing a flap cleavage assay on the target nucleicacid, to thermocycling conditions that include multiple cycles of: afirst temperature of at least 90° C.; a second temperature; and a thirdtemperature in the range of 65° C. to 75° C.; wherein the secondtemperature is at least 10° C. lower than the third temperature; and (b)measuring cleavage of a flap oligonucleotide while the reaction mixtureis at the second temperature.
 15. The method of claim 14, wherein saidsecond temperature is in the range 50° C. to 55° C.
 16. The method ofclaim 14, wherein the measuring step (b) comprises measuring cleavage ofa 5′ fluorophore from the flap oligonucleotide.
 17. The method of claim14, wherein the multiple cycles is 20-50 cycles.
 18. The method of claim17, wherein said method further comprises graphing the amount ofcleavage that occurs in each of the 20-50 cycles, thereby providing anestimate of the abundance of the target nucleic acid.
 19. The method ofclaim 14, wherein the reaction mixture has a volume that is in the rangeof 5 μl to 200 μl, and each of said first to third temperatures is,independently, of a duration in the range of 10 seconds to 3 minutes.20. The method of claim 14, wherein said cleavage of the flapoligonucleotide in (b) indicates a mutation in the target nucleic acid.21. The method of claim 14, wherein: the PCR reagents comprise: anucleic acid template; a first PCR primer, a second PCR primer, athermostable polymerase, and nucleotides; and the flap cleavage reagentscomprise: an invasive oligonucleotide, a flap oligonucleotide, athermostable flap endonuclease and a FRET cassette, wherein i. the firstPCR primer and the invasive oligonucleotide have different nucleotidesequences or ii. the first PCR primer and the invasive oligonucleotidehave the same nucleotide sequence and the first primer is used as theinvasive oligonucleotide
 22. The method of claim 21, wherein said firstPCR primer is present in said reaction mixture at the same concentrationas the second PCR primer.
 23. The method of claim 21, wherein thethermostable polymerase is selected from Taq, Pfu, Pwo, UlTma and Vent.24. The method of claim 14, wherein the target nucleic acid correspondsto a mutated locus in the human genome.
 25. The method of claim 24,wherein the mutated locus is cancer-related.
 26. The method of claim 25,wherein the mutated locus is an oncogenic mutation in PIK3CA, NRAS,KRAS, JAK2, HRAS, FGFR3, FGFR1, EGFR, CDK4, BRAF, RET, PGDFRA, KIT orERBB2.
 27. The method of claim 14, wherein no additional reagents areadded to said reaction mix between steps (a) and (b).
 28. The method ofclaim 14, wherein said reaction mix further comprises PCR reagents andflap reagents for amplifying and detecting a second target nucleic acid.